Methods of treating psychiatric substance abuse, and other disorders using combinations containing omega-3 fatty acids

ABSTRACT

The invention provides methods for treating or preventing psychiatric disorders, substance abuse disorders, and other conditions, e.g., cardiovascular disease and cancer, involving administration of a therapeutically-effective amount of a cytosine-containing or cytidine-containing compound, creatine-containing compound, adenosine-containing, adenosine-elevating compound, omega-3 fatty acids, or combinations thereof to a mammal. The invention further provides methods of enhancing neurodevelopment and delaying premature pregnancy by administration of an effective amount of a cytosine-containing or cytidine-containing compound, creatine-containing compound, adenosine-containing, adenosine-elevating compound, omega-3 fatty acids, or combinations thereof to a mammal.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims benefit of U.S. Provisional Application No.60/509,714, filed Oct. 8, 2003, hereby incorporated by reference.

STATEMENT AS TO FEDERALLY SPONSORED RESEARCH

This invention was funded, in part, by grants DA09448, DA11321, andDA09448, from the National Institute on Drug Abuse, and MH48343,MH53636, and MH63266 from the National Institute of Mental Health. Thegovernment may have certain rights in the invention.

BACKGROUND OF THE INVENTION

This invention relates to compositions and methods for the treatment ofpsychiatric, e.g., depressive, substance abuse, or other disorders.

Psychiatric and substance abuse disorders present unique complicationsfor patients, clinicians, and care givers. These disorders are difficultto diagnose unequivocally and fear of societal condemnation, as well aslack of simple and effective therapies, often results in patients whoare reluctant to disclose their symptoms to health professionals,leading to adverse societal and health consequences.

Psychiatric and substance abuse disorders include alcohol and opiateabuse or dependence, depression, dysthymia, and attention-deficithyperactivity disorder, among others, and occur in people of all agesand backgrounds.

Use of substances such as alcohol and opiates often leads to addictionand dependence on these substances, causing a variety of adverseconsequences, including clinical toxicity, tissue damage, physicaldependence and withdrawal symptoms, and an impaired ability to maintainsocial and professional relationships. The etiology of substance abuseor dependence is unknown, although factors such as the user's physicalcharacteristics (e.g., genetic predisposition, age, or weight),personality, or socioeconomic class have been postulated to bedeterminants.

Depression and dysthymia are prevalent disorders that are often chronicand associated with frequent relapses and long duration of episodes.These disorders include psychosocial and physical impairment and a highsuicide rate among those affected. A lifetime prevalence ofapproximately 17% has been widely reported, and the likelihood ofrecurrence is more than 50% (Angst, J. Clin. Psychiatry 60 Suppl. 6:5-9,1999). Because most antidepressants with clinical efficacy act uponmonoamines (primarily norepinephrine and serotonin), much research ondepression has focused upon interactions between these neurotransmittersand their reuptake transporters and receptor proteins. Mostpharmacotherapies for depression require weeks or months of treatmentdespite immediate effects on brain monoamine transmission. As a result,research has become progressively less focused upon receptors themselvesand more focused upon the intracellular mechanisms of antidepressanttreatments. The neurological mechanisms underlying depression anddysthymia are poorly understood, with a concomitant lack of suitablepharmacological therapies for the treatment of these disorders. Currenttherapies often have many adverse effects and are not suitable foradministration to certain cohorts. For example, depression in theelderly, particularly in those in long-term care facilities, is commonand is often more refractory to treatment than depression in young ormiddle-aged adults; however, the elderly are particularly sensitive tothe common adverse effects of many antidepressant drugs, particularlythe anticholinergic side effects. Similarly, therapies that are suitablefor administration to adults may not be suitable for children.

Attention-deficit hyperactivity disorder (ADHD) is a highly heritableand prevalent neuropsychiatric disorder estimated to affect 6% of theschool-age children in the United States. ADHD typically occurs in earlychildhood and persists into adulthood, but is often not diagnosed untilor after adolescence. Clinical hallmarks of ADHD are inattention,hyperactivity, and impulsivity, which often respond to treatment withstimulants (e.g., methylphenidate, dextroamphetamine, or magnesiumpemoline), although non-stimulant drugs such as beta-blockers (e.g.,propranolol or nadolol), tricyclic antidepressants (e.g., desipramine),and anti-hypertensives (e.g., clonidine) are also used. Treatment withthese drugs, however, is complicated by adverse effects, including thepossibility of abuse of the medication, growth retardation, disturbanceof heart rhythms, elevated blood pressure, drowsiness, depression, sleepdisturbances, headache, stomachache, appetite suppression, reboundreactions, and by the unclear long-term effects of drug administrationon brain function.

Simple and effective pharmacological treatments for these disorders haveproven scarce to date. It would be beneficial to providepharmacotherapies suitable for administration to all populations,including the elderly and children, for the treatment of substance abuseand psychiatric disorders, such as depression.

SUMMARY OF THE INVENTION

In general, the invention features methods of treating psychiatricdisorders, substance abuse or dependency, and other disorders, and theirsymptoms, by administering a cytidine-containing, cytosine-containing,creatine-containing, uridine-containing, adenosine-containing, oradenosine-elevating compound, in combination with an omega-3 fatty acidto a mammal. Substance abuse and dependencies treated by the methodsdescribed herein include, for example, alcohol, opiate, cocaine,amphetamines, methamphetamine, and methylphenidate abuse or dependence.Psychiatric disorders treated by the methods described herein includemood disorders (e.g., unipolar depression, dysthymia, cyclothymia, andbipolar disorder), attention-deficit hyperactivity disorder (ADHD),anxiety disorders (e.g., panic disorder and generalized anxietydisorder), obsessive-compulsive disorder (OCD), post-traumatic stressdisorder (PTSD), phobias, and psychotic disorders (e.g., schizophreniaand schizoaffective disorder). Preferred psychiatric disorders includeunipolar depression, dysthymia, cyclothymia, panic disorder, generalizedanxiety disorder, obsessive-compulsive disorder (OCD), post-traumaticstress disorder (PTSD), and phobias. Other disorders treated by themethods of the invention include cardiovascular disease, cancer,dysmenorrhea, infertility, preeclampsia, postpartum depression,menopausal discomfort, osteoporosis, thrombosis, inflammation,hyperlipidemia, hypertension, rheumatoid arthritis, hyperglyceridemia,and gestational diabetes. In addition, the invention features methods ofenhancing neurodevelopment and delaying premature birth by administeringa cytidine-containing, cytosine-containing, creatine-containing,uridine-containing, adenosine-containing, adenosine-elevating compound,or omega-3 fatty acid to a mammal.

Any of the cytidine-containing, cytosine-containing,creatine-containing, uridine-containing, adenosine-containing,adenosine-elevating compounds, or omega-3 fatty acids of the inventionmay be administered separately or in combination. When a combination ofcompounds is employed, one or more of the compounds may be employed in asubtherapeutically effective amount or an amount insufficient alone toeffect the desired outcome. In this embodiment, the combination isadministered in a therapeutically effective amount or an amountsufficient to effect the desired outcome, even though one or more of theactive ingredients is administered at less than an effective level. Anexemplary combination for use in any of the methods described hereinincludes an omega-3 fatty acid and either a uridine-containing compound,a cytidine-containing compound, or a cytosine-containing compound.

The invention therefore further features compositions including acombination of an omega-3 fatty acid and either a uridine-containingcompound, a cytidine-containing compound, or a cytosine-containingcompound, e.g., wherein at least one compound is present in asubtherapeutically effective amount.

In preferred embodiments of any aspect of the invention, thecytidine-containing compound is cytidine, CDP, or CDP-choline; thecytidine-containing compound includes choline; and the mammal is a humanchild, adolescent, adult, or older adult. In other preferredembodiments, the CDP-choline is administered orally, and theadministration is chronic.

The uridine-containing compound is for example uridine, UMP, UDP, UTP,or triacetyl uridine. Exemplary omega-3 fatty acids includeeicosapentaenoic acid, docosahexaenoic acid, and α-linolenic acid, e.g.,from fish oil, flaxseed oil, or microalgae.

In other preferred embodiments, a brain phospholipid (e.g., lecithin) ora brain phospholipid precursor (e.g., a fatty acid or a lipid), is alsoadministered to the mammal. In other preferred embodiments, anantidepressant is also administered to the mammal.

In other preferred embodiments, the mammal has a co-morbid neurologicaldisease, for example, post-stroke depression.

Treatment methods may also include a diagnosis of the particulardisorder or condition by a physician or other medical professional priorto administration of the particular disorder or condition.Administration of the therapeutic compounds may also occur under thecontinuing care of a physician or medical professional.

As used herein, by “alcohol” is meant a substance containing ethylalcohol. By “opiate” is meant any preparation or derivative of opium,which is a naturally occurring substance extracted from the seed pod ofa poppy plant (e.g., Papaver somniferum) and which contains at least oneof a number of alkaloids including morphine, noscapine, codeine,papaverine, or thebaine. Heroin, an illegal, highly addictive drug isprocessed from morphine. For the purposes of this invention, the termopiate includes opioids.

By “opioid” is meant a synthetic narcotic that resembles an opiate inaction, but is not derived from opium.

By “abuse” is meant excessive use of a substance, particularly one thatmay modify body functions, such as alcohol or opiates.

By “dependency” is meant any form of behavior that indicates an alteredor reduced ability to make decisions resulting, at least in part, fromthe use of a substance. Representative forms of dependency behavior maytake the form of antisocial, inappropriate, or illegal behavior andinclude those behaviors directed at the desire, planning, acquiring, anduse of a substance. This term also includes the psychic craving for asubstance that may or may not be accompanied by a physiologicaldependency, as well as a state in which there is a compulsion to take asubstance, either continuously or periodically, in order to experienceits psychic effects or to avoid the discomfort of its absence. Forms of“dependency” include habituation, that is, an emotional or psychologicaldependence on a substance to obtain relief from tension and emotionaldiscomfort; tolerance, that is, the progressive need for increasingdoses to achieve and sustain a desired effect; addiction, that is,physical or physiological dependence which is beyond voluntary control;and use of a substance to prevent withdrawal symptoms. Dependency may beinfluenced by a number of factors, including physical characteristics ofthe user (e.g., genetic predisposition, age, gender, or weight),personality, or socioeconomic class.

By “dysthymia” or “dysthymic disorder” is meant a chronically depressedmood that occurs for most of the day, more days than not, for at leasttwo years. In children and adolescents, the mood may be irritable ratherthan depressed, and the required minimum duration is one year. Duringthe two year period (one year for children or adolescents), anysymptom-free intervals last no longer than 2 months. During periods ofdepressed mood, at least two of the following additional symptoms arepresent: poor appetite or overeating, insomnia or hypersomnia, lowenergy or fatigue, low self-esteem, poor concentration or difficultymaking decisions, and feelings of hopelessness. The symptoms causeclinically significant distress or impairment in social, occupational(or academic), or other important areas of functioning. The diagnosis ofdysthymia is not made if: the individual has ever had a manic episode, amixed episode, a hypomanic episode; has ever met the criteria for acyclothymic disorder; the depressive symptoms occur exclusively duringthe course of a chronic psychotic disorder (e.g., schizophrenia); or ifthe disturbance is due to the direct physiological effects of asubstance or a general medical condition. After the initial two-years ofdysthymic disorder, major depressive episodes may be superimposed on thedysthymic disorder (“double depression”). (Diagnostic and StatisticalManual of Mental Disorders (DSM IV), American Psychiatric Press, 4^(th)Edition, 1994).

By “unipolar depression” or “major depressive disorder” is meant aclinical course that is characterized by one or more major depressiveepisodes in an individual without a history of manic, mixed, orhypomanic episodes. The diagnosis of unipolar depression is not made if:manic, mixed, or hypomanic episodes develop during the course ofdepression; if the depression is due to the direct physiological effectsof a substance; if the depression is due to the direct physiologicaleffects of a general medical condition; if the depression is due to abereavement or other significant loss (“reactive depression”); or if theepisodes are better accounted for by schizoaffective disorder and arenot superimposed on schizophrenia, schizophreniform disorder, delusionaldisorder, or psychotic disorder. If manic, mixed, or hypomanic episodesdevelop, then the diagnosis is changed to a bipolar disorder. Depressionmay be associated with chronic general medical conditions (e.g.,diabetes, myocardial infarction, carcinoma, and stroke). Generally,unipolar depression is more severe than dysthymia.

The essential feature of a major depressive episode is a period of atleast two weeks during which there is either depressed mood or loss ofinterest or pleasure in nearly all activities. In children andadolescents, the mood may be irritable rather than sad. The episode maybe a single episode or may be recurrent. The individual also experiencesat least four additional symptoms drawn from a list that includeschanges in appetite or weight, sleep, and psychomotor activity;decreased energy; feelings of worthlessness or guilt; difficultythinking, concentrating, or making decisions; or recurrent thoughts ofdeath or suicidal ideation, plans, or attempts. Each symptom must benewly present or must have clearly worsened compared with the person'spreepisode status. The symptoms must persist for most of the day, nearlyevery day, for at least two consecutive weeks, and the episode must beaccompanied by clinically significant distress or impairment in social,occupational (or academic), or other important areas of functioning.(Diagnostic and Statistical Manual of Mental Disorders (DSM IV),American Psychiatric Press, 4^(th) Edition, 1994).

By “neurological disease” is meant a disease, which involves theneuronal cells of the nervous system. Specifically included are priondiseases (e.g., Creutzfeldt-Jakob disease); pathologies of thedeveloping brain (e.g., congenital defects in amino acid metabolism,such as argininosuccinicaciduria, cystathioninuria, histidinemia,homocystinuria, hyperammonemia, phenylketonuria, tyrosinemia, andfragile X syndrome); pathologies of the mature brain (e.g.,neurofibromatosis, Huntington's disease, depression, amyotrophic lateralsclerosis, multiple sclerosis); conditions that strike in adulthood(e.g. Alzheimer's disease, Creutzfeldt-Jakob disease, Lewy body disease,Parkinson's disease, Pick's disease); and other pathologies of the brain(e.g., brain mishaps, brain injury, coma, infections by various agents,dietary deficiencies, stroke, multiple infarct dementia, andcardiovascular accidents). By “co-morbid” or “co-morbidity” is meant aconcomitant but unrelated pathology, disease, or disorder. The termco-morbid usually indicates the coexistence of two or more diseaseprocesses.

By “attention-deficit hyperactivity disorder” or “ADHD” is meant abehavioral disorder characterized by a persistent and frequent patternof developmentally inappropriate inattention, impulsivity, andhyperactivity. Indications of ADHD include lack of motor coordination,perceptual-motor dysfunctions, EEG abnormalities, emotional lability,opposition, anxiety, aggressiveness, low frustration tolerance, poorsocial skills and peer relationships, sleep disturbances, dysphoria, andmood swings (“Attention Deficit Disorder,” The Merck Manual of Diagnosisand Therapy (17^(th) Ed.), eds. M. H. Beers and R. Berkow, Eds., 1999,Whitehouse Station, N.J.).

By “treating” is meant the medical management of a patient with theintent that a cure, amelioration, or prevention of a disease,pathological condition, or disorder will result. This term includesactive treatment, that is, treatment directed specifically towardimprovement of a disease, pathological condition, or disorder, and alsoincludes causal treatment, that is, treatment directed toward removal ofthe cause of the disease, pathological condition, or disorder. Inaddition, this term includes palliative treatment, that is, treatmentdesigned for the relief of symptoms rather than the curing of thedisease, pathological condition, or disorder; preventive treatment, thatis, treatment directed to prevention of the disease, pathologicalcondition, or disorder; and supportive treatment, that is, treatmentemployed to supplement another specific therapy directed toward theimprovement of the disease, pathological condition, or disorder. Theterm “treating” also includes symptomatic treatment, that is, treatmentdirected toward constitutional symptoms of the disease, pathologicalcondition, or disorder.

By “therapeutically-effective amount” is meant an amount of acytidine-containing, cytosine-containing compound, a uridine-containingcompound, a creatine-containing compound, an adenosine-containingcompound, an adenosine-elevating compound, an omega-3 fatty acid, orcombination thereof sufficient to produce a healing, curative,prophylactic, stabilizing, or ameliorative effect in a particulartreatment.

By “subtherapeutically-effective amount” is meant an amount of acytidine-containing, cytosine-containing compound, a uridine-containingcompound, a creatine-containing compound, an adenosine-containingcompound, an adenosine-elevating compound, or omega-3 fatty acid notsufficient on its own to produce a healing, curative, prophylactic,stabilizing, or ameliorative effect in a particular treatment.

By “cytidine-containing compound” is meant any compound that includes,as a component, cytidine, CMP, CDP, CTP, dCMP, dCDP, or dCTP.Cytidine-containing compounds can include analogs of cytidine. Preferredcytidine-containing compounds include, without limitation, CDP-cholineand cytidine 5′-diphosphocholine, frequently prepared as cytidine5′-diphosphocholine [sodium salt] and also known as citicoline.

By “cytosine-containing compound” is meant any compound that includes,as a component, cytosine. Cytosine-containing compounds can includeanalogs of cytosine.

By “adenosine-containing compound” is meant any compound that includes,as a component, adenosine. Adenosine-containing compounds can includeanalogs of adenosine.

By “adenosine-elevating compound” is meant any compound that elevatesbrain adenosine levels, for example, compounds which inhibit or alteradenosine transport or metabolism (e.g., dipyridamole orS-adenosylmethionine).

By “uridine-containing compound” is meant any compound that includes asa component, uridine or UTP. Uridine-containing compounds can includeanalogs of uridine, for example, triacetyl uridine.

By “creatine-containing compound” is meant any compound that includes asa component, creatine. Creatine-containing compounds can include analogsof creatine.

By “phospholipid” is meant a lipid containing phosphorus, e.g.,phosphatidic acids (e.g., lecithin), phosphoglycerides, sphingomyelin,and plasmalogens. By “phospholipid precursor” is meant a substance thatis built into a phospholipid during synthesis of the phospholipid, e.g.,fatty acids, glycerol, or sphingosine.

By “omega-3 fatty acid” is meant a fatty acid having an unsaturated bondthree carbons from the omega carbon. This term encompasses the freeacid, a salt, or an esterified form, e.g., a phospholipid. Omega-3 fattyacids may be mono- or polyunsaturated.

By “child or adolescent” is meant an individual who has not attainedcomplete growth and maturity. Generally, a child or adolescent is undertwenty-one years of age.

By “older adult” is meant an individual who is in the later stage oflife. Generally, an older adult is over sixty years of age.

Unless otherwise stated, all psychiatric and substance abuse disordersare those described in Diagnostic and Statistical Manual of MentalDisorders, 4^(th) ed., Text Revision, Washington, D.C.: AmericanPsychiatric Association, 2000, hereby incorporated by reference.

The present invention provides therapeutics for substance abuse ordependencies, psychiatric disorders, and other disorders and conditions.The compounds utilized herein are relatively non-toxic, and CDP-choline,uridine, triacetyl uridine, and omega-3 fatty acids in particular, arepharmocokinetically understood and known to be well tolerated bymammals. The present invention, therefore, provides treatments that arelikely to have few adverse effects and may be administered to childrenand adolescents, as well as the elderly, or those whose health iscompromised due to existing physical conditions.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a bar graph showing the relative efficacies of CDP-choline andfluoxetine.

FIG. 2 is a graph showing phosphorus-31 MRS data from the human brain.

FIG. 3A is a T1 weighted anatomical image of the basal ganglia andthalamus, indicating regions of interest, used to sample the T2relaxation times, for C (caudate), P (putamen), and T (thalamus).

FIG. 3B is a scatter plot of individual T2 relaxation times for theright putamen of ADHD children treated with placebo and of healthychildren. The increased T2 relaxation times seen in the ADHD sampleindicate diminished regional blood volume.

FIG. 4A is a graph showing the association between T2-RT in rightputamen and accuracy on the performance of the computerized attentiontask for children with ADHD on placebo (closed circles) and normalcontrols (open circles). As indicated there is a significant inverselinear correlation between accuracy and T2 relaxation time (higherlevels of T2-RT indicate lower perfusion).

FIG. 4B is a graph showing the percent change in T2-RT in the rightputamen following treatment with methylphenidate in children with ADHD.Note that the degree of response is affected by the baseline level ofactivity. The higher the temporal scaling the greater the activity ofthe subject. T2-RT change values below zero indicate enhanced regionalblood volume following methylphenidate administration.

FIG. 5 is a schematic illustration of the molecular structure ofCDP-choline.

FIGS. 6A-6C are graphs showing the effects of the standardantidepressant drugs using two separate but complementary methods ofscoring. (A) When latency to become immobile (Mean±SEM) was measured,desipramine (DMI), fluoxetine (FLX) and citalopram (CIT) increasedlatencies to become immobile. (B) When behavioral sampling was used, DMIcaused decreases in occurrences of immobility and increases inoccurrences of climbing, without affecting occurrences of swimming(Means±SEM). This pattern of behaviors is consistent with anoradrenergic mechanism of action (Detke et al. Psychopharmacology121:66-72 1995). In contrast, FLX and CIT decreased immobility andincreased swimming without affecting climbing, a pattern of behaviorsconsistent with a serotonergic mechanism of action (Detke et al.Psychopharmacology 121:66-72 1995). (C) The antidepressant drugs did notaffect the weights of the rats. *P 0.05, **P<0.01, Fisher's HSD tests,7-12 rats per group.

FIGS. 7A-7C are graphs showing the effects of uridine (URI) alone onbehaviors in the FST. (A) URI dose-dependently increased latencies tobecome immobile. (B) URI dose-dependently decreased immobility andincreased swimming without affecting climbing, a pattern of behaviorssimilar to that seen with SSRIs such as FLX and CIT. (C) URI did notaffect the weights of the rats. *P<0.05, **P<0.01, Fisher's HSD tests,7-12 rats per group.

FIGS. 8A-8E are graphs showing the effects of dietary supplementationwith omega-3 fatty acids (OMG) on behaviors in the FST. During the firstexposure to forced swimming, OMG supplementation had no effect onlatencies to become immobile (A) or behavior subtypes (B), regardless ofthe length of pre-exposure. During the re-test, however, OMGexposure-dependently increased latencies to become immobile (C). OMGalso exposure-dependently decreased immobility and increased swimmingwithout affecting climbing (D), a pattern of behaviors similar to thatseen with SSRIs. (E) The OMG treatments did not affect the weights ofthe rats. *P<0.05, **P<0.01, Fisher's HSD tests, 7-12 rats per group.

FIG. 9A-9E are graphs showing the effects of a normallysubtherapeutically effective dose of URI (71.7 mg/kg) in rats thatreceived normally subtherapeutically effective dietary supplementationwith OMG (3 or 10 days) on behaviors in the FST. As expected, OMGsupplementation had no effect on latencies to become immobile (A) orbehavior subtypes (B) during the first exposure to forced swimming.During the re-test, however, this low dosage of URI increased latenciesto become immobile (C) in rats given 10 but not 3 days of OMGsupplementation. This low dosage of URI also decreased immobility andincreased both swimming and climbing (D) in rats given 10 but not 3 daysof OMG supplementation. This pattern of behaviors is different from thatseen with TCAs or SSRIs. (E) Combined treatment with URI and OMG did notaffect the weights of the rats. *P<0.05, **P<0.01, Fisher's HSD tests,7-12 rats per group.

FIGS. 10A and 10B are graphs showing the effects of treatments withantidepressant-like efficacy in the FST on locomotor activity in ratsgiven one exposure to forced swimming. (A) None of the treatmentsaffected behavior when distance traveled in an open field (Mean±SEM, incm) rather than swimming was measured during re-testing. (B) The weightsof the rats did not differ among these treatments. *P<0.05, **P<0.01,Fisher's HSD tests, 6-8 rats per group.

DETAILED DESCRIPTION OF THE INVENTION

The invention described herein features compositions and methods for thetreatment of substance abuse disorders, such as alcohol and opiate abuseor dependence, psychiatric disorders, such as mood disorders (e.g.,unipolar depression, dysthymia, cyclothymia, and bipolar disorder),attention-deficit hyperactivity disorder (ADHD), anxiety disorders(e.g., panic disorder and generalized anxiety disorder),obsessive-compulsive disorder (OCD), post-traumatic stress disorder(PTSD), phobias, and psychotic disorders (e.g., schizophrenia andschizoaffective disorder), and their symptoms, and other disorders, suchas cardiovascular disease, cancer, dysmenorrhea, infertility,preeclampsia, postpartum depression, menopausal discomfort,osteoporosis, thrombosis, inflammation, hyperlipidemia, hypertension,rheumatoid arthritis, hyperglyceridemia, and gestational diabetes. Theinvention also features methods for enhancing neurodevelopment anddelaying premature birth.

For these indications, the invention features the use ofcytidine-containing, cytosine-containing, uridine-containing,creatine-containing, adenosine-containing, or adenosine-elevatingcompounds or omega-3 fatty acids. A preferred cytidine-containingcompound is CDP-choline (also referred to as citicoline or CDP choline[sodium salt]), a preferred adenosine-containing compound isS-adenosylmethionine (SAMe), and a preferred uridine-containing compoundis triacetyl uridine.

The cytidine-containing, cytosine-containing, uridine-containing,creatine-containing, adenosine-containing, or adenosine-elevatingcompounds may be co-administered with other compounds that areprecursors for the synthesis of brain phospholipids, e.g., fatty acids(such as omega-3 fatty acids), lipids, or lecithin.

When combinations of the therapeutic agents described herein, e.g., anomega-3 fatty acid and uridine, are employed, unexpected synergisticeffects are observed. Such combinations enable the use of asubtherapeutically effective amount of one or more of the components ofthe combination to achieve a therapeutic effect.

Mood Disorders

Alterations in brain phospholipid metabolism may be involved in thepathophysiology of mood disorders such as depression, bipolar disorder,dysthymia, and cyclothymia. Because phospholipid metabolism affects thefluidity of neural membranes, it can play a critical role inextracellular processes including surface receptor binding andmembrane-protein interactions, as well as intracellular processesincluding signal transduction and mitochondrial function (Pacheco et al.Prog Neurobiol 50:255-273 1996; Shetty et al. J Neurochem 67: 1702-17101996; Exton Eur J Biochem 243:10-20 1997; Nomura et al. Life Sci68:2885-2891 2001). Depression has been linked to abnormalities in bothmembrane synthesis and fluidity (Moore et al. American Journal ofPsychiatry 154:116-118 1997; Sonawalla et al. Am J Psychiatry156:1638-1640 1999; Detke et al. Archives of General Psychiatry57:937-943 2000; Moore et al. Bipolar Disorder 3:207-216 2000; Steingardet al. Biol Psychiatry 48:1053-1061 2000). Treatments that affect themetabolism of phospholipids or their incorporation into neural membranesmay therefore have efficacy in the treatment of depression and othermood disorders.

Although there is evidence that treatments that affect phospholipidmetabolism and membrane fluidity have some efficacy in the treatment ofdepressive symptoms, the effects are often modest and causalrelationships are difficult to prove. For example, populations withdiets rich in fish show lower prevalences of major depression (HibbelnLancet 351:1213, 1998). Fish is particularly high in omega-3 fattyacids, which are long-chain polyunsaturated fatty acids that areincorporated into neuronal membranes (for review, see Freeman Ann ClinPsychiatry 12:159-165, 2000). The double bonds within polyunsaturatedfatty acids such as omega-3 fatty acids result in structuralconformations that prevent dense packing of phospholipids, therebyinfluencing membrane fluidity (Popp-Snijders et al. Scand J Clin LabInvest. 44: 39-46, 1984; Cartwright et al. Atherosclerosis 55:267-281,1985). Treatment with omega-3 fatty acids in humans decreases brainwater proton transverse relaxation times (T2s), consistent withincreased membrane fluidity. Although omega-3 fatty acids have not beenevaluated in controlled clinical trials of major depression, theyimprove the course of illness in patients with bipolar disorder, whichinvolves depressive states (Stoll et al. Arch Gen Psychiatry 56:407-412, 1999). Similarly, some symptoms of cocaine withdrawal, whichoften involves depressive symptoms, can be treated in clinicalpopulations with citicoline (Renshaw et al. Psychopharmacology142:132-138, 1999). Citicoline is metabolized in part to the nucleosidecytidine, which induces the biosynthetic pathways of structural membranephospholipids and increases membrane production (Lopez-Coviella et al. JNeurochem 65:889-894, 1995; Knapp et al. Brain Res 822:52-59, 1999).Short-term administration of cytidine by systemic injection hasantidepressant-like effects in rats (Carlezon et al. Biol. Psychiatry51:882-889, 2002). Cytidine is further converted to the nucleosideuridine (Wurtman et al. Biochem Pharmacol 60:989-992, 2000), but neitherof these agents has been examined in clinical studies of patients withmood disorders.

We have now discovered that CDP-choline is efficacious in human trialsand that cytidine-containing and cytosine-containing compounds can beused to treat depression. CDP-choline has been found to have twoimportant new therapeutic properties. First, CDP-choline improves brainchemistry, e.g., increases phospholipid synthesis, in healthy adults.This effect is particularly apparent in older adults. Second,CDP-choline has antidepressant effects that are similar to those offluoxetine, a widely-used drug for the treatment of depression.

Cytidine-containing and cytosine-containing compounds are particularlyefficacious in treating the elderly, and these compounds are efficaciousin treating depression in patients with a co-morbid neurological disease(e.g., post-stroke depression). In addition, these compounds may beadministered in conjunction with, and thereby work synergistically with,phospholipids (e.g., lecithin) or compounds that are precursors for thesynthesis of brain phospholipids (e.g., fatty acids or lipids).

We have now also discovered that uridine and omega-3 fatty acids areefficacious, alone and in combination, in a treatment for unipolardepression or dysthymia. The therapeutic properties ofuridine-containing compounds are similar to those of cytidine-containingcompounds, while omega-3 fatty acids appear to produce an increase inmembrane fluidity. In addition, the combination of a uridine-containingcompound and an omega-3 fatty acid produces a synergistic effect, i.e.,the combination of the two agents requires a reduced dose of eachconstituent.

Substance Abuse or Dependence

Phosphorus-31 magnetic resonance spectroscopy (MRS) studies indicatethat persons who are dependent upon alcohol and opiates have decreasedbrain levels of phospholipids. In addition, data derived from healthyolder persons, indicates that chronic administration of CDP-choline isassociated with neurochemical changes consistent with phospholipidsynthesis. Increasing brain levels of cytosolic adenosine also provideseffective therapy for alcohol or opiate abuse or dependency, becauseenergy in the form of ATP is required to support phospholipid synthesis.Based on our results described herein, omega-3 fatty acids are utilizedin another method of the invention to treat substance abuse ordependence, e.g., from alcohol, opiates, cocaine, amphetamines,methamphetamine, and methylphenidate. Omega-3 fatty acids may also beused in combination with other compounds as described herein.

Attention Deficit Hyperactivity Disorder (ADHD)

Functional magnetic resonance imaging (fMRI) experiments in childrendiagnosed with ADHD indicate that symptoms of hyperactivity andinattention are strongly correlated with measures of blood flow withinthe putamen nuclei, which are strongly dopaminergic brain regions. Inaddition, administration of methylphenidate, a stimulant used to treatADHD, increases blood flow in the putamen in parallel with a decrease inmotor activity. ADHD symptoms may be closely tied to functionalabnormalities in the putamen, which is predominantly involved in theregulation of motor behavior. Accordingly, because cytidine-containingand cytosine-containing compounds (e.g., CDP-choline) have dopaminergicactivity, these compounds may be used to treat persons diagnosed withADHD without many of the side effects associated with stimulanttherapies. In particular, treatments with cytidine-containing orcytosine-containing compounds are effective in treating hyperactivity inchildren diagnosed with ADHD. Based on our results described herein,ADHD may also be treated with uridine-containing compounds, or acombination including an omega-3 fatty acid and either acytidine-containing, cytosine-containing, uridine-containing,creatine-containing, adenosine-containing, or adenosine-elevatingcompound (e.g., a uridine-containing compound or a cytidine-containingcompound), or a combination thereof.

Other Psychiatric Disorders

Omega-3 fatty acids may be used in the treatment of other psychiatricdisorders, such as anxiety disorders (e.g., panic disorder andgeneralized anxiety disorder) obsessive-compulsive disorder (OCD),post-traumatic stress disorder (PTSD), phobias, and psychotic disorders(e.g., schizophrenia and schizoaffective disorder). In these treatments,omega-3 fatty acids may be used in combination with acytidine-containing, cytosine-containing, uridine-containing,creatine-containing, adenosine-containing, or adenosine-elevatingcompound.

Neurodevelopment

The compounds of the invention may also be employed to enhanceneurodevelopment, e.g., neurite growth. Exemplary combinations for thisindication include an omega-3 fatty acid and a cytidine-containing,cytosine-containing, uridine-containing, creatine-containing,adenosine-containing, or adenosine-elevating compound. Methods forevaluating the enhancement of neurodevelopment are known in the art(e.g., Gibson, R. A. and M. Makrides Acta Paediatr, 1998, 87:1017-22,Fewtrell, M. S., et al., J Pediatr, 2004, 144:471-9, Fewtrell, M. S., etal., Pediatrics, 2002, 110:73-82, O'Connor, D. L., et al., Pediatrics,2001, 108:359-71, Clandinin, M., et al., Pediatric Res, 2002,51:187A-8A, Innis, S. M., et al., J Pediatr, 2002, 140:547-54,Clandinin, M. T., et al., Pediatr Res, 1997, 42:819-25, Uauy, R., etal., J Pediatr, 1994, 124:612-20, Werkman, S. H. and S. E. Carlson,Lipids, 1996, 31:91-7., Carlson, S. E., et al., Eur J Clin Nutr, 1994,48 Suppl 2:S27-30., Vanderhoof, J., et al, J Pediatr Gastroenterol Nutr,2000, 31:121-7, and Marszalek, J. R., et al., J Biol Chem, 2004,279:23882-91). Exemplary methods for gauging neurodevelopment includethe Bayley Mental Developmental Index (MDI), the Bayley PsychomotorDevelopmental Index (PDI), Knobloch, Passamanick and Sherrard'sDevelopmental Screening Inventory, and the Fagan Test of InfantIntelligence. Enhancement can be measured, for example, relative to acontrol group, such as a group that did not receive the compounds of theinvention.

Cardiovascular Disease

The compounds of the invention may also be employed to treatcardiovascular disease (CVD), including atherosclerosis, coronary arterydisease, regression and decreased progression of coronary lesions,decrease in triglyceride blood levels, increase in HDL cholesterol,neutralization of LDL cholesterol, reduction in mortality from cardiacevents, and decrease in ventricular tachycardia. Exemplary combinationsfor these indications include an omega-3 fatty acid and acytidine-containing, cytosine-containing, uridine-containing,creatine-containing, adenosine-containing, or adenosine-elevatingcompound.

Oncology

The compounds of the invention may also be employed to treat cancer,including reducing the risk of developing cancer (Larsson, S. C., etal., Am J Clin Nutr, 2004, 79:935-45), treating cancer cachexia duringradio and chemotherapy and increasing the rate of recovery (Heller, A.R., et al., Int J Cancer, 2004, 111:611-6), and treatingcancer-associated wasting (Jatoi, A., et al., J Clin Oncol, 2004,22:2469-76). Exemplary cancers include breast, colon, pancreatic,chronic myelogenous leukemic, and melanoma. Exemplary combinations forthese indications include an omega-3 fatty acid and acytidine-containing, cytosine-containing, uridine-containing,creatine-containing, adenosine-containing, or adenosine-elevatingcompound.

Women's Health

The methods of the invention also address a number of medical problemsthat exclusively or particularly effect women, e.g., dysmenorrhea,infertility (e.g., by increasing uterine blood flow), preeclampsia,postpartum depression, menopausal discomfort, and osteoporosis. Thecompounds of the invention may also be employed to delay prematurebirth, e.g., by balancing eicosanoids involved in labor and improvingplacental blood flow. Exemplary combinations for these indicationsinclude an omega-3 fatty acid and a cytidine-containing,cytosine-containing, uridine-containing, creatine-containing,adenosine-containing, or adenosine-elevating compound.

Other Indications

The compounds of the inventions may also be used treat otherindications, such as thrombosis, inflammation, hyperlipidemia,hypertension, rheumatoid arthritis, hyperglyceridemia, and gestationaldiabetes. Exemplary combinations for these indications include anomega-3 fatty acid and cytidine-containing, cytosine-containing,uridine-containing, creatine-containing, adenosine-containing, oradenosine-elevating compounds.

Cytidine-Containing and Cytosine-Containing Compounds

Useful cytidine-containing or cytosine-containing compounds may includeany compound including one of the following: cytosine, cytidine, CMP,CDP, CTP, dCMP, dCDP, and dCTP. Preferred cytidine-containing compoundsinclude CDP-choline and cytidine 5′-diphosphocholine [sodium salt]. Thislist of cytidine-containing and cytosine-containing compounds isprovided to illustrate, rather than to limit the invention, and thecompounds described above are commercially available, for example, fromSigma Chemical Company (St. Louis, Mo.).

CDP-choline is a naturally occurring compound that is hydrolyzed intoits components of cytidine and choline in vivo. CDP-choline issynthesized from cytidine-5′-triphosphate and phosphocholine withaccompanying production of inorganic pyrophosphate in a reversiblereaction catalyzed by the enzyme CTP:phosphocholine cytidylyltransferase(Weiss, Life Sciences 56:637-660, 1995). CDP-choline is available fororal administration in a 500 mg oblong tablet. Each tablet contains522.5 mg CDP-choline sodium, equivalent to 500 mg of CDP-choline.Matching placebo tablets are also available. The excipients contained inboth active and placebo tablets are talc, magnesium stearate, colloidalsilicon dioxide, hydrogenated castor oil, sodiumcarboxy-methylcellulose, and microcrystalline cellulose. The molecularstructure of CDP-choline [sodium salt] is provided in FIG. 5.

Other formulations for treatment or prevention of psychiatric andsubstance abuse disorders may take the form of a cytosine-containing orcytidine-containing compound combined with a pharmaceutically-acceptablediluent, carrier, stabilizer, or excipient.

Adenosine-Containing and Adenosine-Elevating Compounds

Adenosine-containing or adenosine-elevating compounds provide usefultherapies because these compounds provide the ATP needed forphospholipid synthesis. Useful adenosine-containing oradenosine-elevating compounds include, without limitation, any compoundcomprising one of the following adenosine, ATP, ADP, or AMP. Onepreferred adenosine-containing compound is S-adenosylmethionine (SAMe).

In addition, compounds are known that are capable of increasingadenosine levels by other mechanisms. For example, adenosine uptake canbe inhibited by a number of known compounds, including propentofylline(described in U.S. Pat. No. 5,919,789, hereby incorporated byreference). Another known compound that inhibits adenosine uptake isEHNA.

Other useful compounds that can be used to increase brain adenosinelevels are those that inhibit enzymes that break down adenosine, (e.g.,adenosine deaminase and adenosine kinase). Finally, administeringcompounds that contain adenosine or precursors of adenosine, which arereleased as adenosine in vivo, can also be used.

Uridine-Containing Compounds

Uridine and uridine-containing compounds may provide useful therapiesbecause these compounds can be converted to CTP, a rate-limiting factorin PC biosynthesis (Wurtman et al., Biochemical Pharmacology 60:989-992,2000). Useful uridine-containing compounds include, without limitation,any compound comprising uridine, UTP, UDP, or UMP. Uridine anduridine-containing compounds and analogs are well tolerated in humans.The oral bioavailability of uridine in humans can be increased byvarious means, e.g., acetylation of ring hydroxyl groups as in triacetyluridine. Alternatively, formulations may be used to increasebioavailbility.

Creatine-Containing Compounds

Creatine and creatine-containing compounds provide useful therapiesbecause these compounds, by virtue of increasing brain phospholipidlevels, can raise the levels of ATP. Creatine and creatine-containingcompounds are known to be well tolerated at relatively high doses inhumans.

Omega-3 Fatty Acids

Omega-3 fatty acids provide useful therapy likely because they increasemembrane fluidity. Exemplary omega-3 fatty acids includeeicosapentaenoic acid, docosahexaenoic acid, and α-linolenic acid.Omega-3 fatty acids may be administered as the free acid, a salt, or inesterified form (e.g., as triglycerides or phospholipids). Omega-3 fattyacids may be obtained in pure form by synthesis or by culture ofmicroalgae. Omega-3 fatty acids may also be administered in a mixturefrom a naturally occurring source, e.g., fish oil, flaxseed oil,soybeans, rapeseed oil, or microalgae. The use of omega-3 fatty acidswith other therapeutic compounds of the invention may produce asynergistic effect, i.e., the combination of the two agents requires areduced dose of each constituent.

Administration

Conventional pharmaceutical practice is employed to provide suitableformulations or compositions for administration to patients. Oraladministration is preferred, but any other appropriate route ofadministration may be employed, for example, parenteral, intravenous,subcutaneous, intramuscular, intracranial, intraorbital, ophthalmic,intraventricular, intracapsular, intraspinal, intracistemal,intraperitoneal, intranasal, or aerosol administration. Therapeuticformulations may be in the form of liquid solutions or suspensions (as,for example, for intravenous administration); for oral administration,formulations may be in the form of liquids, tablets, or capsules; andfor intranasal formulations, in the form of powders, nasal drops, oraerosols. In particular, omega-3 fatty acids may be administered in aninclusion complex, dispersion (such as a micelle, microemulsion, andemulsion), or liposome, for example, as described in U.S. applicationSer. No. ______, titled “ENHANCED EFFICACY OF OMEGA-3 FATTY ACID THERAPYIN THE TREATMENT OF PSYCHIATRIC DISORDERS,” filed on Oct. 8, 2004. Inaddition, compounds useful in the methods described herein also includeencapsulated compounds, e.g., liposome- or polymer-encapsulatedcytidine-containing, cytosine-containing, uridine-containing,creatine-containing, adenosine-containing, and adenosine-elevatingcompounds. Useful compounds further include those linked (e.g.,covalently or non-covalently) to various antibodies, ligands, or othertargeting and enveloping or shielding agents (e.g., albumin ordextrose), to allow the cytidine-containing, cytosine-containing,uridine-containing, creatine-containing, adenosine-containing, oradenosine-elevating compound to reach the target site (e.g., the centralnervous system) prior to being removed from the blood stream, e.g., bythe kidneys and liver, and prior to being degraded.

Methods well known in the art for making formulations are described, forexample, in Remington: The Science and Practice of Pharmacy (20th ed.)ed. A. R. Gennaro, Lippincott: Philadelphia 2003. Formulations forparenteral administration may, for example, contain excipients, sterilewater, saline, polyalkylene glycols such as polyethylene glycol, oils ofvegetable origin, or hydrogenated naphthalenes.

If desired, slow release or extended release delivery systems may beutilized. Biocompatible, biodegradable lactide polymer,lactide/glycolide copolymer, or polyoxyethylene-polyoxypropylenecopolymers may be used to control the release of the compounds. Otherpotentially useful parenteral delivery systems include ethylene-vinylacetate copolymer particles, osmotic pumps, implantable infusionsystems, and liposomes. Formulations for inhalation may containexcipients, for example, lactose, or may be aqueous solutionscontaining, for example, polyoxyethylene-9-lauryl ether, glycocholateand deoxycholate, or may be oily solutions for administration in theform of nasal drops, or as a gel.

Preferably, the compounds of the invention, such as CDP-choline, areadministered at a dosage of at least 500 mg twice daily by oraladministration. Orally administered CDP-choline is bioavailable, withmore than 99% of CDP-choline and/or its metabolites absorbed and lessthan 1% excreted in feces. CDP-choline, administered either orally orintravenously, is rapidly converted into the two major circulatingmetabolites, choline and cytidine. Major excretion routes are lung(12.9%) and urine (2.4%); the rest of the dose (83.9%) is apparentlymetabolized and retained in tissues.

In general, the compounds of the invention, such as CDP-choline,uridine, UTP, creatine, or SAMe, are administered at a dosageappropriate to the effect to be achieved and are typically administeredin unit dosage form. The dosage preferably ranges from 50 mg per day to2000 mg per day. The exact dosage of the compound may be dependent, forexample, upon the age and weight of the recipient, the route ofadministration, and the severity and nature of the symptoms to betreated. In general, the dosage selected should be sufficient toprevent, ameliorate, or treat a particular indication, or one or moresymptoms thereof, or effect a particular outcome without producingsignificant toxic or undesirable side effects. As noted above, thepreferred route of administration for most indications is oral.

In the case of CDP-choline, there have been no reported cases ofoverdoses. CDP-choline toxicity is largely self-limiting, ingestion oflarge amounts in preclinical studies shows common cholinergic symptoms(salivation, lacrimation, urination, defecation, and vomiting).

Combination with Other Therapeutics

The cytidine-containing, cytosine-containing, uridine-containing,creatine-containing, adenosine-containing, adenosine-elevatingcompounds, and omega-3 fatty acids of the invention may be administeredas a monotherapy, in combination with each other, or in combination withother medicaments for the indications described herein.

Preferably, the compounds of the invention may be administered inconjunction with lower doses of current medicaments for theseindications, including stimulants and antidepressants. For example, thecompounds of the invention may be administered with phospholipids, e.g.,lecithin, or with brain phospholipid precursors, e.g., fatty acids orlipids, or may be administered as an adjunct to standard therapy for thetreatment of psychiatric or substance abuse disorders.

In one particular example, the compound of the invention may beadministered in combination with an antidepressant, anticonvulsant,antianxiety, antimanic, antipyschotic, antiobsessional,sedative-hypnotic, stimulant, or anti-hypertensive medication. Examplesof these medications include, but are not limited to, the antianxietymedications, alprazolam, buspirone hydrochloride, chlordiazepoxide,chlordiazepoxide hydrochloride, clorazepate dipotassium, desipraminehydrochloride, diazepam, halazepam, hydroxyzine hydrochloride,hydroxyzine pamoate, lorazepam, meprobamate, oxazepam, prazepam,prochlorperazine maleate, prochlorperazine, prochlorperazine edisylate,and trimipramine maleate; the anticonvulsants, amobarbital, amobarbitalsodium, carbamazepine, chlordiazepoxide, chlordiazepoxide hydrochloride,clorazepate dipotassium, diazepam, divalproex sodium, ethosuximide,ethotoin, gabapentin, lamotrigine, magnesium sulfate, mephenyloin,mephobarbital, methsuximide, paramethadione, pentobarbital sodium,phenacemide, phenobarbital, phenobarbital sodium, phensuximide,phenyloin, phenyloin sodium, primidone, secobarbital sodium,trimethadione, valproic acid, and clonazepam; the antidepressants,amitriptyline hydrochloride, amoxapine, bupropion hydrochloride,clomipramine hydrochloride, desipramine hydrochloride, doxepinhydrochloride, fluoxetine, fluvoxamine, imipramine hydrochloride,imipramine pamoate, isocarboxazid, lamotrigine, maprotolinehydrochloride, nortriptyline hydrochloride, paroxetine hydrochloride,phenelzine sulfate, protriptyline hydrochloride, sertralinehydrochloride, tranylcypromine sulfate, trazodone hydrochloride,trimipramine maleate, and venlafaxine hydrochloride; the antimanicmedications, lithium carbonate and lithium citrate; the antiobsessionalmedications, fluvoxamine, and clomipramine hydrochloride; theantipsychotic medications, acetophenazine maleate, chlorpromazinehydrochloride, chlorprothixene, chlorprothixene hydrochloride,clozapine, fluphenazine decanoate, fluphenazine enathrate, fluphenazinehydrochloride, haloperidol decanoate, haloperidol, haloperidol lactate,lithium carbonate, lithium citrate, loxapine hydrochloride, loxapinesuccinate, mesoridazine besylate, molindone hydrochloride, perphenazine,pimozide, prochlorperazine maleate, prochlorperazine, prochlorperazineedisylate, promazine hydrochloride, risperidone, thioridazine,thioridazine hydrochloride, thiothixene, thiothixene hydrochloride, andtrifluoperzine hydrochloride; the sedative-hypnotic medications,amobarbital, amobarbital sodium, aprobarbital, butabarbital, chloralhydrate, chlordiazepoxide, chlordiazepoxide hydrochloride, clorazepatedipotassium, diazepam, diphenhydramine, estazolam, ethchlorvynol,flurazepam hydrochloride, glutethimide, hydroxyzine hydrochloride,hydroxyzine pamoate, lorazepam, methotrimeprazine hydrochloride,midazolam hydrochloride, oxazepam, pentobarbital sodium, phenobarbital,phenobarbital sodium, quazepam, secobarbital sodium, temazepam,triazolam, and zolpidem tartrate; the stimulants, dextroamphetaminesulfate, methamphetamine hydrochloride, methylphenidate hydrochloride,and pemoline; and the anti-hypertensive, clonidine.

The following examples are provided for the purpose of illustrating theinvention and should not be construed as limiting.

Unipolar Depression or Dysthymia

Treatment of Human Subjects with Cytidine- or Cytosine-ContainingCompounds

Proton and phosphorus magnetic resonance (MR) spectroscopy studies ofsubjects with mood disorders have characterized two patterns of alteredneurochemistry associated with depression. The first pattern indicates achange (increase or decrease) in cytosolic choline, as well as increasedfrontal lobe phosphomonoesters, while the second pattern points todecreased brain purines (cytosolic adenosine-containing compounds) anddecreased nucleoside triphosphates (NTP). The former results reflectaltered phospholipid metabolism, while the latter results indicatechanges in cerebral energetics. Although few longitudinal studies havebeen conducted, these altered metabolite levels appear to be mood state,rather than trait, dependent.

To assess whether chronic CDP-choline administration leads to detectablechanges in lipid metabolite resonances in phosphorus-31 MR spectra,eighteen healthy subjects (mean age: 70) were administered 500 mg of anoral formulation of CDP-choline daily for a six week period. From weeks6 to 12, half of the subjects continued to receive CDP-choline and halfreceived placebo in a double-blind fashion. The MR data demonstratedthat CDP-choline treatment was associated with a significant increase inbrain phosphodiesters (p=0.008), a finding that is indicative ofincreased phospholipid synthesis. Neuropsychological testing alsorevealed increases in verbal fluency (p=0.07), verbal learning(p=0.003), visuospatial learning (p=0.0001) across all subjects at weektwelve. CDP-choline administration, therefore, improves measures ofverbal fluency and spatial memory in healthy adults and results inincreased brain phospholipid synthesis in older adults, particularlyduring chronic administration.

In a second study, twelve depressed subjects (mean age 40) received 500mg of an oral formulation of CDP-choline twice daily for eight weeks.With eight weeks of treatment, mean 17-item Hamilton Depression RatingScale (HDRS) scores decreased from 21±3 to 10±7 (p<0.0001). A successfulresponse to CDP-choline was also associated with a reduction in theproton MR spectroscopic cytosolic choline resonance in the anteriorcingulate cortex. Comparable data for forty-one depressed subjectsparticipating in imaging trials and treated with open label fluoxetine,20 mg/day for eight weeks, demonstrated reductions in HDRS scores from21±4 to 11±6 (p<0.0001) (FIG. 1). CDP-choline and fluoxetine wereassociated with complete responses in 6/12 (50%) and 17/41 (41%) of thesubjects, respectively (FIG. 1). In depressed adults, therefore, theantidepressant effects of CDP-choline were comparable to those offluoxetine.

These data represent the first demonstration that human brain lipidmetabolism can be modified using pharmacological strategies, and that,particularly in older adults, treatment is associated with improvedcognitive performance. These data demonstrate that therapeuticstrategies, using cytosine- and cytidine-containing compounds (e.g.,CDP-choline), that are aimed at reversing biochemical alterations arebeneficial for the treatment of depression or dysthymia.

Use of Citicoline in a Rodent Model of Depression

The effects of citicoline were examined in the forced swim test (FST), arodent model of depression as described herein. Because citicoline israpidly converted to cytidine and choline, their effects were alsoexamined in the FST. Citicoline did not have antidepressant effects inrats in the FST over a range of doses (50-500 mg/kg, IP) shown to haveneuroprotective effects in experimental ischemia in rodents. In fact,high doses of citicoline appeared to have small pro-depressant effectsin this model. Molar equivalent amounts of cytidine (23.8-238 mg/kg, IP)had significant antidepressant effects in the FST, whereas molarequivalent amounts of choline (13.7-136.6 mg/kg, IP) had significantpro-depressant effects. The optimally effective dose of cytidine (238mg/kg, IP) did not affect locomotor activity or establish conditionedrewarding effects at therapeutic concentrations.

Use of Uridine and Omega-3 Fatty Acids in a Rat Model of Depression

The behavioral effects of the combination of uridine and omega-3 fattyacids were also evaluated in rats using the forced swim test (FST). Thisassay identifies in rodents treatments that have antidepressant effectsin humans (Porsolt et al. Nature 266:730-732 1977; Carlezon et al. Biol.Psychiatry 51:882-889, 2002). Uridine was administered using systemicinjection while omega-3 fatty acids were administered by supplementationwithin the diet for various periods of time (3, 10, or 30 days). Theeffects of uridine in rats maintained on the omega-3 fatty acid-enricheddiet were also evaluated to determine if these effects were additive.For comparison, the effects of the standard antidepressant drugsdesipramine (a tricyclic antidepressant [TCA]) and fluoxetine andcitalopram (selective serotonin reuptake inhibitors [SSRIs]) weredetermined. The efficacy of each treatment in the FST was evaluatedusing two separate scoring methods: latency to become immobile, a simpleand rapid method that identifies agents with antidepressant effects(Pliakas et al. J Neurosci 21:7397-7403 2001), and behavioral sampling,a more complex method that differentiates antidepressant drugs accordingto their pharmacological mechanisms (Detke et al. Psychopharmacology121:66-72 1995). Finally, treatments with antidepressant-like effects inthe FST were evaluated for non-specific effects on activity in an openfield, which might complicate interpretation of the data from theswimming studies.

Methods

Rats: A total of 197 male Sprague-Dawley rats (Charles RiverLaboratories, Boston Mass.) were used in these studies. The rats werehoused in groups of four and weighed 325-375 gm at the time ofbehavioral testing. Rats were maintained on a 12 h light (0700-1900h)-12 h dark cycle with free access to food and water except duringtesting. Experiments were conducted in accordance with the 1996 Guidefor the Care and Use of Laboratory Animals (NIH) and McLean Hospitalpolicies.

Drugs: Dosages of desipramine HCl (DMI), fluoxetine HCl (FLX),citalopram HBr (CIT), and uridine (URI) were administered in a distilledwater vehicle (VEH) at a volume of 1 cc/kg. All drugs were purchasedfrom RBI-Sigma (St. Louis, Mo.) except CIT, which was a gift of ForestLaboratories (New York, N.Y.). Fatty acids were administered as adietary supplement in food fortified with either menhaden oil (OMG)containing omega-3 fatty acids, or olive oil (CON), as a control, eachat 4.5% w/w (Research Diets Inc., New Brunswick N.J.). The menhaden oilcontained 27% w/w omega-3 fatty acids, and the rats ate an average of 25gm of food (0.3 gm OMG) each day. The diets were equivalent in overallfat, protein, carbohydrate, and caloric content.

Forced Swim Test (FST): One hundred-sixty seven rats were used in theFST studies, which were conducted as described previously (Carlezon etal. Biol. Psychiatry 51:882-889, 2002) with minor modifications. The FSTis a two-day procedure in which rats swim under conditions in whichescape is not possible. On the first day, rats are placed in clear, 65cm tall-25 cm diameter cylinders filled to 48 cm with 25° C. water. Therats initially struggle to escape from the water, but eventually theyadopt a posture of immobility in which they make only the movementsnecessary to keep their heads above water. After 15 min of forcedswimming, the rats are removed from the water, dried with towels, andplaced in a warmed enclosure for 30 min. The cylinders are emptied andcleaned between rats. When the rats are re-tested 24 hours later underidentical conditions in 5 min sessions, immobility is increased.Treatment with standard antidepressant drugs within the 24 hr periodbetween the first exposure to forced swimming and re-testing canattenuate facilitated immobility, an effect correlated withantidepressant efficacy in humans (Porsolt et al. Nature 266:730-7321977; Detke et al. Psychopharmacology 121:66-72 1995, Carlezon et al.Biol. Psychiatry 51:882-889, 2002).

Rats tested with DMI, FLX, CIT, or URI received 3 separateintraperitoneal (IP) injections of drug (or VEH), at 1 hr, 19 hr, and 23hr after the first exposure to forced swimming. This commonly usedregimen is sensitive to the antidepressant-like effects of many standardagents (Porsolt et al. Nature 266:730-732 1977; Detke et al.Psychopharmacology 121:66-72 1995; Carlezon et al. Biol. Psychiatry51:882-889, 2002). Rats tested with OMG (or CON) received the specialdiets 3, 10, or 30 days prior to the start of the swim test, andreceived saline or URI injections (IP) at 1, 19, and 23 hr after theforced swim. There were 7-12 rats per treatment condition, and separaterats were used for each treatment regimen.

Swim tests were videotaped from the side of the cylinders, and scored byraters unaware of the treatment conditions. The re-test (day 2) of theFST was videotaped for the groups receiving only DMI, FLX, CIT, URI, orVEH injections because these rats had not received any treatments beforethe first exposure to forced swimming. Both days of FST testing werevideotaped for rats that were maintained on the special diets becausethe groups differed before the first exposure to forced swimming. Ratswere scored using two separate but complementary methods: latency toimmobility and behavioral sampling. Latency to become immobile wasdefined as the time at which the rat first initiated a stationaryposture that did not reflect attempts to escape from the water. In thischaracteristic posture, the forelimbs are motionless and tucked towardthe body. To qualify as immobility, this posture had to be clearlyvisible and maintained for >2.0 sec. For behavioral sampling, rats wererated at 5 sec intervals throughout the duration of the forced swimmingsession. At each 5 sec interval, the predominant behavior was assignedto one of 4 categories: immobility, swimming, climbing, or diving (Detkeet al. Psychopharmacology 121:66-72 1995). A rat was judged to beimmobile if it was making only movements necessary to keep its headabove water, climbing if it was making forceful thrashing movements withits forelimbs directed against the walls of the cylinder, swimming if itwas actively making swimming movements that caused it to move within thecenter of the cylinder, and diving if it swam below the water, towardthe bottom of the cylinder. Diving behavior rarely occurred, and it wasnot affected by any of the treatments tested. The behavioral samplingmethod reportedly differentiates classes of antidepressant drugs: forexample, TCAs decrease immobility and increase climbing withoutaffecting swimming, whereas SSRIs decrease immobility and increaseswimming without affecting climbing (Detke et al. Psychopharmacology121:66-72 1995).

Data from the tests with the standard agents (DMI, FLX and CIT) wereanalyzed together, whereas data from the tests with URI alone wereanalyzed separately. For these treatments, latencies to become immobileor the number of occurrences of each category of behavior was analyzedusing separate one-way (treatment) analyses of variance (ANOVAs).Significant effects were analyzed further using post hoc Fisher'shonestly significant difference (HSD) tests. Data from the tests withOMG alone and OMG plus URI were analyzed separately, and each day oftesting was analyzed independently. For these treatment regimens,latencies to become immobile or the number of occurrences of eachcategory of behavior was analyzed using separate two-way(treatment×duration of diet) analyses of variance (ANOVAs), followed bypost hoc Fisher's HSD tests.

Locomotor activity: Thirty rats were used to determine if the treatmentsthat were effective in the FST studies had non-specific effects onactivity levels in rats exposed previously to forced swimming. Thesestudies were conducted exactly as the FST studies had been conducteduntil the time of re-testing: that is, all rats underwent the first dayof the FST, but 24 hr later they were placed for 1 hr in automated,17×17×12 in (L×W×H) open field activity chambers (Med Associates, St.Albans Vt.) instead of being re-exposed to forced swimming. There were6-8 rats per treatment condition; control rats received injections ofVEH. The total distance traveled (in cm) during the test session wasquantified, and data were analyzed with a one-way (treatment) ANOVAfollowed by post hoc Fisher's HSD tests. The researchers who establishedthe FST interpreted the facilitated immobility during the secondexposure to forced swimming as an indicator of “behavioral despair,” adepressive-like symptom (Porsolt et al. Nature 266:730-732, 1977).Regardless of the etiology of facilitated immobility, all of the majorclasses of antidepressant treatments—including TCAs, SSRIs, atypicals,monoamine oxidase inhibitors, and electroconvulsive shock therapy(Porsolt et al. Nature 266:730-732, 1977; Borsini et al. Psychopharmacol94:147-160, 1988; Detke et al. Psychopharmacology 121:66-72,1995)—effectively reduce indicators of immobility in the FST. Indeed,the main strength of the FST is its ability to identify, in rats,treatments with antidepressant efficacy in people (WillnerPsychopharmacology 83:1-16, 1984). DMI, FLX and CIT reduced immobilitywhen given by injection within the time between the first and secondexposure to forced swimming. A similar treatment regimen with uridinealso reduced indicators of immobility in the FST, indicating that thisagent has antidepressant-like effects in rats. Rats fed a diet enrichedwith omega-3 fatty acids were also less immobile in the FST, consistentwith antidepressant-like effects. A normally sub-effective dose ofuridine had antidepressant-like effects in rats given a normallysub-effective treatment regimen of dietary supplementation with omega-3fatty acids, suggesting that the antidepressant-like effects of thesetwo treatments can potentiate one another. Considered together, thesedata provide strong evidence in an animal model that treatments thataffect phospholipid metabolism and membrane fluidity may have promise inthe treatment of depressive-like symptoms in humans.

Results

Standard antidepressant treatments (DMI, FLX, CIT) reduced indicators ofimmobility in the FST during the re-test (day 2), regardless of thescoring method that was used. These agents affected latencies to becomeimmobile (F_(3,39)=5.73, P<0.01) when this method of scoring was used(FIG. 6A): the amount of time that elapsed before the first bout ofimmobility was increased by DMI (10 mg/kg; P<0.01, Fisher's HSD), FLX(20 mg/kg; P<0.05) and CIT (5.0 mg/kg; P<0.01). These agents alsoaffected the patterns of behavior when the sampling method was used(FIG. 6B): they caused differences in the number of occurrences ofimmobility (F_(3,39)=9.14, P<0.01), swimming (F_(3,39)=10.3, P<0.01),and climbing (F_(3,39)=16.1, P<0.01) behaviors. Consistent with previousobservations (Detke et al. Psychopharmacology 121:66-72, 1995), DMI (aTCA) reduced immobility and increased climbing (P's<0.01) withoutaffecting swimming, whereas FLX and CIT (SSRIs) reduced immobility andincreased swimming (P's<0.01) without affecting climbing. The weights ofthe rats did not differ among groups at the time of the re-test (FIG.6C), which is important because weight can influence swimming behaviors(Pliakas et al. J Neurosci 21:7397-7403, 2001).

URI had dose-dependent effects on latencies to become immobile(F_(3,32)=3.05, P 0.05) (FIG. 7A): this agent increased latencies at 239mg/kg (P<0.05), but not at 130 or 71.7 mg/kg. With the behavioralsampling method (FIG. 7B), URI significantly affected the occurrences ofimmobility (F_(3,32)=3.10, P<0.05) and swimming (F_(3,32)=3.07, P<0.05)without affecting climbing. URI reduced immobility (P<0.01) andincreased swimming behaviors (P<0.01) at 239 mg/kg only. This pattern ofbehaviors is similar to that seen after treatment with SSRIs. Theweights of the rats did not differ among groups at the time of there-test (FIG. 7C).

The effects of dietary supplementation with OMG alone depended upon thelength of treatment, and were apparent only during the re-test session.During the first exposure to forced swimming, dietary OMG had no effecton latencies to become immobile (FIG. 8A) or any of the behaviorsubtypes (FIG. 8B). During the re-test, however, OMG affected latenciesto become immobile (Main effect of treatment: F_(1,50)=4.08, P<0.05)(FIG. 8C): latencies were elevated in rats that had received OMG for 30days (P<0.05), but not for 10 or 3 days. Similarly, OMG significantlyaffected occurrences of immobility (treatment×duration interaction:F_(1,50)=3.22, P<0.05) and swimming (treatment×duration interaction:F_(1,50)=3.42, P<0.05) without affecting climbing (FIG. 8D). OMG reducedimmobility (P<0.01) and increased swimming behaviors (P 0.01) after 30days treatment only. This pattern of behaviors is similar to that seenafter treatment with SSRIs. The weights of the rats did not differbetween treatment groups at the time of the re-test (FIG. 8E).

Administration of a sub-effective dosage of URI affected behavior inrats maintained on a normally sub-effective regimen of OMG dietarysupplementation. Confirming earlier observations, OMG supplementationfor 3 or 10 days had no effects on behaviors during the first exposureto forced swimming (FIG. 9A-9B). During the re-test, however, latenciesto become immobile were altered in OMG-fed rats that also received 71.7mg/kg URI (Main effect of duration: F_(1,28)=4.52, P<0.05) (FIG. 9C):latencies were elevated in rats that had received URI after OMGsupplementation for 10 days (P<0.05), but not for 3 days. Likewise, thecombination of normally sub-effective treatments with URI and OMGaffected immobility (Main effect of treatment: F_(1,28)=17.7, P<0.01),swimming (Main effect of treatment: F_(1,28)=6.46, P<0.02), and climbing(treatment×duration interaction: F_(1,28)=7.77, P<0.01) behaviors (FIG.9D). URI treatment reduced immobility (P<0.01), increased swimming(P<0.05) and increased climbing (P<0.05) in rats given 10 days, but not3 days, of OMG. The weights of the rats did not differ between treatmentgroups at the time of the re-test (FIG. 9E).

None of the treatments with antidepressant-like effects in the FSTaffected activity levels when rats were tested in open field chambersrather than the forced swim cylinders during the re-test (FIG. 10A). Theweights of the rats did not differ among these groups (FIG. 10B).

The FST in rats is a useful model for predicting beneficial effects oftherapies for depression in humans. The effects of uridine in the FSTare similar to those for equimolar concentrations of cytidine. Themechanisms by which uridine and cytidine have antidepressant-likeeffects in the FST are unknown. One possibility is that thesenucleosides affect the synthesis or fluidity of neural membranes(Lopez-Coviella et al. J Neurochem 65:889-894, 1995; Knapp et al., 1999;Wurtman et al. Biochem Pharmacol 60:989-992, 2000), each of which may beanomalous in mood disorders (Moore et al. American Journal of Psychiatry154:116-118 1997; Sonawalla et al. Am J Psychiatry 156:1638-1640 1999;Detke et al. Archives of General Psychiatry 57:937-943 2000; Moore etal. Bipolar Disorder 3:207-216 2000; Steingard et al. Biol Psychiatry48:1053-1061 2000). Another possibility is that the actions of uridineare mediated through its ability to alter catecholamine function in thebrain. While the effects of uridine per se on catecholamine function arenot known, citicoline increases brain production of neurotransmitterssuch as norepinephrine and dopamine, possibly by affecting precursorssuch as tyrosine (Martinet et al. Arch Int Pharmacodyn 239: 52-56 1979).To begin exploring the mechanisms by which uridine hasantidepressant-like effects, we scored the FST using behavioralsampling, a detailed scoring method that can differentiate betweenvarious classes of antidepressant agents (Detke et al.Psychopharmacology 121:66-72 1995). Consistent with previous studies inwhich behavioral sampling was used (Detke et al. Psychopharmacology121:66-72 1995), the standard norepinephrine uptake inhibitordesipramine decreased measures of immobility and increased measures ofclimbing without affecting measures of swimming. Conversely, thestandard SSRIs fluoxetine, and citalopram decreased immobility andincreased swimming without affecting climbing. Although differentialeffects on the swimming and climbing measures may involve factors otherthan norepinephine-serotonin interactions, the effects of uridine in theFST resemble those of fluoxetine and citalopram (altered immobility andswimming) rather than those of desipramine (altered immobility andclimbing) indicating that uridine may be effective in this assay becauseof effects on serotonergic function.

The mechanisms by which omega-3 fatty acids have antidepressant-likeeffects are unknown. Omega-3 fatty acids appear to have profound effectson the fluidity of neural membranes. Importantly, theantidepressant-like effects of omega-3 fatty acids were seen only withlong-term dietary enrichment, and not after shorter regimens. Theseresults may explain the subtle effects of omega-3 fatty acids in humans,and highlight the challenges that complicate clinical studies with thistype of agent. Furthermore, the effects were not seen in the rats duringthe first exposure to forced swimming, but only during the re-test.Inasmuch as facilitated immobility in the FST is due to activation ofintracellular signaling pathways and genes associated with stress(Pliakas et al. J Neurosci 21:7397-7403, 2001), these findings suggestthat omega-3 fatty acids interfere with the induction ofneuroadaptations that contribute to development of immobility behaviorsthat may reflect learned helplessness.

Treatment with low dosages of uridine made shorter treatment regimens ofomega-3 fatty acids effective in the FST. Although the mechanisms ofthis interaction are unknown, it seems likely that the effects ofnucleosides on membrane synthesis (Lopez-Coviella et al. J Neurochem65:889-894, 1995; Knapp et al., 1999; Wurtman et al. Biochem Pharmacol60:989-992, 2000) may facilitate the incorporation of omega-3 fattyacids into neural membranes, where they can affect extracellularprocesses including surface receptor binding and membrane-proteininteractions, as well as intracellular processes including signaltransduction and mitochondrial function (Pacheco et al. Prog Neurobiol50:255-273 1996; Shetty et al. J Neurochem 67: 1702-1710 1996; Exton EurJ Biochem 243:10-20 1997; Nomura et al. Life Sci 68:2885-2891 2001). Theeffects on membrane fluidity may be particularly important withinmitochondria, which are vital for energy metabolism and have a highconcentration of polyunsaturated fatty acids within their innerphospholipid membranes (Buttriss et al. Biochim Biophys Acta 962:81-90,1988; Raederstorff et al. Lipids 26:781-787, 1991). Indeed,dysregulation of mitochondria, function is suspected indepression-related syndromes such as bipolar disorder (Kato et al.Bipolar Disorder 2:180-190, 2000), and individuals with bipolar disorderappear to benefit from omega-3 fatty acid therapy (Stoll et al. Arch GenPsychiatry 56: 407-412, 1999).

Alcohol or Opiate Abuse or Dependence

Measurement of Brain Phospholipids

The broad component within the phosphorus-31 MR spectrum, arising fromhuman brain phospholipids, may be measured reliably (FIG. 2).Preliminary results indicate that in persons with alcohol and/or opiatedependence, the intensity of this broad phospholipid resonance isdecreased by 10-15% relative to values for comparison subjects.Accordingly, therapeutic strategies that are aimed at reversing thisbiochemical alteration, for example, by increasing phospholipidsynthesis, are beneficial for the treatment of alcohol and/or opiatedependence.

CDP-choline Administration Leads To Increased Phospholipid Synthesis

To assess whether chronic CDP-choline administration leads to detectablechanges in lipid metabolite resonances in phosphorus-31 MR spectra,eighteen healthy subjects (mean age: 70) were administered 500 mg of anoral formulation of CDP-choline daily for a six week period. From weeks6 to 12, half of the subjects continued to receive CDP-choline and halfreceived placebo in a double-blind fashion. The MR data demonstratedthat CDP-choline treatment was associated with a significant increase inbrain phosphodiesters (p=0.008), a finding that is indicative ofincreased phospholipid synthesis. Neuropsychological testing alsorevealed increases in verbal fluency (p=0.07), verbal learning(p=0.003), visuospatial learning (p=0.0001) across all subjects at weektwelve. CDP-choline administration, therefore, improves measures ofverbal fluency and spatial memory in healthy adults and results inincreased brain phospholipid synthesis in older adults, particularlyduring chronic administration.

Attention Deficit Hyperactivity Disorder (ADHD)

Functional Magnetic Resonance Imaging of Children Diagnosed with ADHD

A new fMRI procedure (T2 relaxometry or “T2-RT”) was developed toindirectly assess blood volume in the striatum (caudate and putamen) ofboys 6-12 years of age under steady-state conditions. Six healthycontrol boys (10.2±1.5 yr) and eleven boys diagnosed with ADHD (9.3±1.6yr) served as subjects in the study to examine fMRI differences betweenunmedicated healthy controls and ADHD children on either placebo or thehighest dose of methylphenidate. The healthy controls were screenedusing structured diagnostic interview (K-SADS-E; Orvaschel, H. &Puig-Antich, J., The schedule for affective disorders and schizophreniafor school-age children-epidemiologic version (Kiddie-SADS-E),University of Pittsburgh, Pittsburgh, Pa., 1987), were free of any majorpsychiatric disorder, and had no more than 3 out of 9 possible symptomsof inattention or hyperactivity-impulsivity by DSM-IV criteria. Childrenwith ADHD were included if they met criteria for ADHD on structureddiagnostic interview, and had at least 6 of 9 symptoms of inattention orhyperactivity-impulsivity. Children with ADHD took part in a tripleblind parent, child, rater), randomized, placebo-controlled study ofeffects of methylphenidate (0, 0.5, 0.8, 1.5 mg/kg in divided dose) onactivity, attention, and fMRI. Children with ADHD were treatedcontinuously for one week with placebo or a specific dose ofmethylphenidate and at the end of the week were tested for drug efficacyusing objective measures of attention and activity and fMRI (SeeMethods) within 1-3 hours of their afternoon dose. The time between doseand testing was held constant for each subject throughout the fourtreatment conditions. Activity and attention were evaluated inunmedicated healthy controls using the same procedure as children withADHD, and fMRI followed within the same time frame.

T2 relaxometry, a novel fMRI procedure, was used to derive steady stateblood flow measures and to test for enduring medication effects.Although conventional Blood Oxygenation Level Dependent (BOLD) fMRI is avaluable technique for observing dynamic brain activity changes betweenbaseline and active conditions, thus far it has failed to provideinsight into possible resting or steady-state differences in regionalperfusion between groups of subjects, or to delineate effects of chronicdrug treatment on basal brain function. T2 relaxometry, like BOLD,hinges on the paramagnetic properties of deoxyhemoglobin. However, themismatch between blood flow and oxygen extraction that occurs as anacute reaction to enhanced neuronal activity in BOLD does not persistunder steady state conditions. Instead, regional blood flow is regulatedto appropriately match perfusion with ongoing metabolic demand, anddeoxyhemoglobin concentration becomes constant between regions in thesteady-state. Therefore, regions with greater continuous activity areperfused at a greater rate, and these regions receive, over time, agreater volume of blood and a greater number of deoxyhemoglobinmolecules per volume of tissue. Thus, there is an augmentation in theparamagnetic properties of the region that is detectable as a diminishedT2 relaxation time.

Conventional T2-weighted images provide only a rough estimate of T2,useful for identifying areas of pathology with markedly different T2properties, such as tumors. To calculate T2-RT with sufficient accuracyto be able to reliably perceive small (ca. 2%) differences in T2 of graymatter associated with functional changes in blood volume, we used fastechoplanner imaging to establish a signal intensity decay curve based on32 sequential measures at different echo times. For each of the 32images, a refocused spin echo was observed.

Highly accurate laboratory-based measures of activity and attention wereobtained by having the children perform a computerized vigilance testwhile an infrared motion analysis system captured and recorded movements(see Methods). These findings were used to ascertain associationsbetween regional measures of T2-RT and capacity to inhibit motoractivity to low levels while attending to a monotonous but demandingtask.

As expected, boys with ADHD on placebo did not sit as still as healthycontrols during the attention tests. They spent more time moving(temporal scaling: F_(1,14)=9.42, P=0.008) and had less complex movementpatterns (spatial scaling: F_(1,14)=9.68, P=0.008). On the continuousperformance task (CPT), a measure of attention, children with ADHD wereless accurate (92.0% vs. 97.1%; F_(1,14)=2.94, P=0.10), and had a morevariable response latency (F_(1,14)=3.11, P<0.10), though thesedifferences did not reach statistical significance in this limitedsample.

Differences in the caudate and putamen regions of children with ADHD andhealthy controls, as well as the change in the T2-RT in these regions inresponse to methylphenidate, were also studied by imaging. The thalamuswas evaluated as a contrast region in which group differences or drugeffects were not expected. No significant differences emerged betweenADHD children on placebo and healthy controls in bilateral T2-RTmeasures for the caudate nucleus (F_(1,14)=2.80, P=0.12). In contrast,ADHD children and controls differed markedly in bilateral putamen T2-RTmeasures (77.9±1.1 msec vs. 76.1±1.1 msec; F_(1,14)=9.40, P=0.008). Onaverage, T2-RT was 3.1% higher in ADHD children than in controls in theleft putamen (F_(1,14)=14.5, P=0.002; FIG. 3B) and 1.6% higher in theright (F_(1,14)=2.62, P=0.13).

For healthy controls and ADHD children on placebo, there were marked andsignificant correlations between motor activity and T2-RT for theputamen bilaterally, but not for caudate or thalamus (Table 1A).Temporal scaling and average time spent immobile, two measures ofactivity-inactivity, correlated −0.752 (P<0.001) and −0.730 (P<0.001),respectively with T2-RT in putamen. The complexity of the movementpattern also correlated with T2-RT in putamen (r_(s)=0.630, P<0.01).Similarly, in unilateral analyses, all three motor activity measurescorrelated with T2 measures for both right and left putamen (Table 1A).

There were also robust correlations between measures of CPT performanceand T2-RT in the putamen bilaterally (Table 1B). Accuracy on the CPTcorrelated −0.807 (P<0.0001) with T2-RT, while variability (S.D.) inresponse latency correlated 0.652 (P<0.005). These associations wereobserved in both right and left putamen (Table 1B, FIG. 4A). Inaddition, there was also a significant association between accuracy onthe CPT task and T2-RT for right, but not left, thalamus. As indicatedin FIG. 4A, there is a significant inverse linear correlation betweenaccuracy and T2 relaxation time (higher levels of T2-RT indicate lowerperfusion).

Methylphenidate exerted robust effects on attention, enhancingperformance accuracy (F_(1,10)=5.98, P<0.05) and reducing responsevariability (S.D.) from 242 to 149 msec (F_(1,10)=14.5, P<0.005).Methylphenidate also exerted significant effects on activity, producinga 126% increase in time spent immobile (F_(1,10)=5.47, P<0.05), andincreasing the complexity of the movement pattern (F_(1,10)=5.73,P<0.05). However, drug effects on activity were strongly dependent onthe subject's unmedicated activity level. For instance, spatialcomplexity increased 52.6% in the 6 subjects who were objectivelyhyperactive (at least 25% more active than normal controls) on placebo(F_(1,5)=13.16, P<0.02), but was unaffected (<8% increase) in the 5 ADHDchildren who were not (p>0.6).

T2-RT in both right and left putamen were significantly altered byongoing treatment with methylphenidate (ANCOVA: F_(1,9)=12.81, P=0.006),although the response was strongly tied to the subject's unmedicatedactivity state (Drug×temporal scaling covariant F_(1,9)=11.09, P=0.008;FIG. 4B). Methylphenidate failed to exert significant effects on T2-RTin thalamus (F_(1,9)=0.13, P>0.7). A trend-level difference was observedin the right caudate (F_(1,9)=3.85 P=0.08).

Overall, as higher T2-RT corresponds to lower perfusion, the presentfindings of increased T2-RT in the putamen of children with ADHD, andthe correlation between T2-RT and objective markers of disease severity,are consistent with some earlier studies. Furthermore, the presentfindings also suggest that a considerable proportion of the variancebetween subjects in degree of hyperactivity and inattention can beaccounted for by T2-RT differences within the putamen alone.

In summary, boys with ADHD (n=11) had higher T2 relaxation time (T2-RT)measures in putamen bilaterally than healthy controls (n=6; P=0.008).Relaxation times correlated with the child's capacity to sit still(r_(s)=−0.75, P<0.001), and his accuracy in performing a computerizedattention task (r_(s)=−0.81, P<0.001). Blinded, placebo-controlled dailytreatment with methylphenidate significantly altered T2-RT in theputamen of children with ADHD (P=0.006), though the magnitude anddirection of the effect was strongly dependent on the child'sunmedicated activity state. A similar but non-significant trend wasobserved in the right caudate. T2-RT measures in the thalamus did notdiffer significantly between groups, and were not affected bymethylphenidate.

Methods

Assessment of Activity and Attention. Activity and attention data werecollected as previously described (Teicher et al., J. Am. Acad. ChildAdolesc. Psychiatry 35: 334-342, 1996). In brief, children sat in frontof a computer and were evaluated using a simple GO/NO-GO CPT in whichthe subject responds to visual presentation of a target and withholdsresponse to a non-target stimuli that appear in the center of the screenat a fixed 2 second inertial interval (Greenberg et al.,Psychopharmacol. Bull. 23: 279-282,1987). The stimuli are simplegeometric shapes that can be distinguished without right/leftdiscrimination, and are designed to allow children with dyslexia toperform as well as normal controls. Three 5-minute test sessions wererecorded during a 30-minute test period while an infrared motionanalysis system (Qualisys, Glastonbury, Conn.) recorded the movement ofsmall reflective markers attached to the head, shoulder, elbow, and backof the child. The motion analysis system stored the precise vertical andhorizontal position of the centroid of each marker 50 times per secondto a resolution of 0.04 mm.

Results were analyzed using the concept of “micro-events.” A newmicro-event begins when the marker moves 1.0 millimeters or more fromits most recent resting location, and is defined by its position andduration. The spatial scaling exponent is a measure of the spatialcomplexity of the movement path, and is calculated from the logarithmicrate of information decay at progressively lower levels of resolution.The temporal scaling exponent is a scale invariant stochastic measure ofpercent time active. Values range from 0 (immobility) to 1 (incessantactivity), and are calculated from the slope of the log-log relationshipbetween the duration of micro-events and their frequency (Paulus et al.,Neuropsychopharmacology 7:15-31, 1992). Software for presenting stimuli,recording activity, and analyzing results was written by M. Teicher andlicensed to Cygnex Inc.

T2 Relaxometry fMRI Procedure and Relaxation Time Computations.

Children were positioned in the scanner and instructed to remain asstill as possible. Images were acquired using a 1.5-T magnetic resonancescanner (Signa, General Electric Medical Systems, Milwaukee, Wis.)equipped with a whole-body, resonant gradient set capable of echo planarimaging (Advanced NMR Systems, Inc., Wilmington, Mass.), and a standardquadrature head coil for image detection. During each examination, 3categories of images were obtained: (1) Scout images (typicallyT1-weighted sagittal images); (2) High resolution T1-weighted matchedaxial images through the ten planes for which maps of T2 were generated;and (3) 32 spin echo, echoplanar image sets, with TE incremented by 4msec in each consecutive image set (e.g., TE (1)=32 msec, TE (2)=36msec, . . . TE (32)=160 msec) through the same ten axial planes (TR=10sec, Slice thickness=7 mm with a 3 mm skip, in-plane resolution=3.125mm×3.125 mm, FOV=200 mm). The 32 TE-stepped images were then transferredto an off-line workstation and corrected for in plane motion using amodification of the DART image registration algorithm (Maas et al.,Magn. Reson. Med. 37:131-139, 1997). The value of T2-RT was thenestimated on a pixel-wise basis by linear regression of the signalintensity S(x,y,n) assuming an exponential decay of S(x,y,n) with timeconstant T2-RT(x,y), such that In S(x,y,TE(n))=1nS(x,y,TE=0)−(TE(n)/T2-RT(x,y)), where (x,y) is the pixel position andTE(n) is the spin-echo time corresponding to the nth image of theseries.

Calculations of regional T2-RT were made for left and right anteriorcaudate, putamen, and thalamus (as a contrast region) using anatomicboundaries observed in T1 weighted images and conservativelycircumscribed to avoid encroaching into ventricular space (see FIG. 3Afor regions of interest). Delineation of regions and analysis of imagingdata was performed on coded images, and the responsible researcher wasblind to the identity, diagnosis, or treatment condition of the subject.T2-RT was calculated from the median value of all the designated pixels,as the median provides a regional estimate less susceptible tocontamination by spurious values from bordering white matter andcerebrospinal fluid regions than the mean.

The intrinsic reliability of the T2-RT measure was determined using awithin subject procedure with head repositioning when necessary. Therewas a lag between end of the first session and start of the secondsession of ca. 5 minutes. Based on 8 within-session comparisons withnormal adult volunteers we observed a correlation of 0.942, and anaverage mean value difference of −0.17% for T2-RT of the putamen.

Statistical Analyses. Differences between groups was assessed usingANCOVA with age as a covariate. Although the groups did not differsignificantly in age, the behavioral and fMRI measures showedage-dependent changes, and ANCOVA minimized this component of the errorvariance. Correlations were calculated using Spearman Rank-Order test.Differences between behavioral and fMRI measures of ADHD subjects onmethylphenidate vs. placebo were assessed using repeated measure ANCOVAwith placebo activity (temporal scaling) as a covariate. This wascrucial in the analysis, as methylphenidate effects are stronglyrate-dependent, and basal activity on placebo accounted for ca. 50% ofthe magnitude of the medication effect.

Other Embodiments

All publications and patent applications mentioned in this specificationare herein incorporated by reference to the same extent as if eachindependent publication or patent application was specifically andindividually indicated to be incorporated by reference.

While the invention has been described in connection with specificembodiments thereof, it will be understood that it is capable of furthermodifications and this application is intended to cover any variations,uses, or adaptations of the invention following, in general, theprinciples of the invention and including such departures from thepresent disclosure that come within known or customary practice withinthe art to which the invention pertains and may be applied to theessential features hereinbefore set forth, and follows in the scope ofthe appended claims.

Other embodiments are within the appended claims.

1. A method of treating a psychiatric disorder in a mammal, said methodcomprising administering to said mammal a combination comprising (i) acytidine-containing compound, cytosine-containing compound, or auridine-containing compound and (ii) an omega-3 fatty acid, wherein saidcombination is administered in a therapeutically effective amount. 2.The method of claim 1, wherein said a cytidine-containing compound,cytosine-containing compound, uridine-containing compound, or omega-3fatty acid is administered in a subtherapeutically effective amount. 3.The method of claim 1, wherein said uridine-containing compound isuridine, UTP, or triacetyl uridine.
 4. The method of claim 1, whereinsaid cytidine-containing compound is cytidine or CDP.
 5. The method ofclaim 1, wherein said cytidine-containing compound further comprisescholine.
 6. The method of claim 1, wherein said cytidine-containingcompound is CDP-choline.
 7. The method of claim 1, wherein said omega-3fatty acid is eicosapentaenoic acid, docosahexaenoic acid, orα-linolenic acid.
 8. The method of claim 1, wherein said omega-3 fattyacid is administered as fish oil, flaxseed oil, or microalgae.
 9. Themethod of claim 1, wherein said psychiatric disorder is a mood disorder10. The method of claim 10, wherein said mood disorder is bipolardisorder, unipolar depression, cyclothymia, or dysthymia.
 11. The methodof claim 1, wherein said psychiatric disorder is attention deficithyperactivity disorder
 12. The method of claim 1, wherein saidpsychiatric disorder is obsessive-compulsive disorder (OCD),post-traumatic stress disorder (PTSD), or a phobia.
 13. The method ofclaim 1, wherein said psychiatric disorder is a psychotic disorder. 14.The method of claim 13, wherein said psychotic disorder is schizophreniaor schizoaffective disorder.
 15. The method of claim 1, wherein saidpsychiatric disorder is an anxiety disorder.
 16. The method of claim 15,wherein said anxiety disorder is panic disorder or generalized anxietydisorder.
 17. A method of treating substance abuse or dependency in amammal, said method comprising administering to said mammal acombination comprising (i) a cytidine-containing compound,cytosine-containing compound, creatine-containing compound,uridine-containing compound, adenosine-containing compound, oradenosine-elevating compound and (ii) an omega-3 fatty acid, whereinsaid combination is administered in a therapeutically effective amount.18. The method of claim 17, wherein said cytidine-containing compound,cytosine-containing compound, creatine-containing compound,uridine-containing compound, adenosine-containing compound, oradenosine-elevating compound, or omega-3 fatty acid is administered in asubtherapeutically effective amount.
 19. The method of claim 17, whereinsaid compound in (i) is a cytidine-containing compound,cytosine-containing compound, or uridine-containing compound.
 20. Themethod of claim 17, wherein said uridine-containing compound is uridine,UTP, or triacetyl uridine.
 21. The method of claim 17, wherein saidcytidine-containing compound is cytidine or CDP.
 22. The method of claim17, wherein said cytidine-containing compound further comprises choline.23. The method of claim 17, wherein said cytidine-containing compound isCDP-choline.
 24. The method of claim 17, wherein said omega-3 fatty acidis eicosapentaenoic acid, docosahexaenoic acid, or α-linolenic acid. 25.The method of claim 17, wherein said omega-3 fatty acid is administeredas fish oil or flaxseed oil.
 26. The method of claim 17, wherein saidsubstance is alcohol or an opiate.
 27. The method of claim 17, whereinsaid substance is cocaine, amphetamines, methamphetamine, ormethylphenidate.
 28. A method of treating cardiovascular disease,cancer, dysmenorrhea, infertility, preeclampsia, postpartum depression,menopausal discomfort, osteoporosis, thrombosis, inflammation,hyperlipidemia, hypertension, rheumatoid arthritis, hyperglyceridemia,or gestational diabetes in a mammal, said method comprisingadministering to said mammal a combination comprising (i) acytidine-containing compound, cytosine-containing compound,creatine-containing compound, uridine-containing compound,adenosine-containing compound, or adenosine-elevating compound and (ii)an omega-3 fatty acid, wherein said combination is administered in atherapeutically effective amount.
 29. The method of claim 28, whereinsaid cytidine-containing compound, cytosine-containing compound,creatine-containing compound, uridine-containing compound,adenosine-containing compound, or adenosine-elevating compound, oromega-3 fatty acid is administered in a subtherapeutically effectiveamount.
 30. The method of claim 28, wherein said compound in (i) is acytidine-containing compound, cytosine-containing compound, oruridine-containing compound.
 31. The method of claim 28, wherein saiduridine-containing compound is uridine, UTP, or triacetyl uridine. 32.The method of claim 28, wherein said cytidine-containing compound iscytidine or CDP.
 33. The method of claim 28, wherein saidcytidine-containing compound further comprises choline.
 34. The methodof claim 28, wherein said cytidine-containing compound is CDP-choline.35. The method of claim 28, wherein said omega-3 fatty acid iseicosapentaenoic acid, docosahexaenoic acid, or α-linolenic acid. 36.The method of claim 28, wherein said omega-3 fatty acid is administeredas fish oil or flaxseed oil.
 37. A method of enhancing neurodevelopmentin a mammal, said method comprising administering to said mammal acombination comprising (i) a cytidine-containing compound,cytosine-containing compound, creatine-containing compound,uridine-containing compound, adenosine-containing compound, oradenosine-elevating compound and (ii) an omega-3 fatty acid, whereinsaid combination is administered in an amount effective to enhanceneurodevelopment.
 38. The method of claim 37, wherein saidcytidine-containing compound, cytosine-containing compound,creatine-containing compound, uridine-containing compound,adenosine-containing compound, or adenosine-elevating compound, oromega-3 fatty acid is not administered in an amount effective to enhanceneurodevelopment.
 39. The method of claim 37, wherein said compound in(i) is a cytidine-containing compound, cytosine-containing compound, oruridine-containing compound.
 40. The method of claim 37, wherein saiduridine-containing compound is uridine, UTP, or triacetyl uridine. 41.The method of claim 37, wherein said cytidine-containing compound iscytidine or CDP.
 42. The method of claim 37, wherein saidcytidine-containing compound further comprises choline.
 43. The methodof claim 37, wherein said cytidine-containing compound is CDP-choline.44. The method of claim 37, wherein said omega-3 fatty acid iseicosapentaenoic acid, docosahexaenoic acid, or α-linolenic acid. 45.The method of claim 37, wherein said omega-3 fatty acid is administeredas fish oil or flaxseed oil.
 46. A method of delaying premature birth ina mammal, said method comprising administering to said mammal acombination comprising (i) a cytidine-containing compound,cytosine-containing compound, creatine-containing compound,uridine-containing compound, adenosine-containing compound, oradenosine-elevating compound and (ii) an omega-3 fatty acid, whereinsaid combination is administered in an amount effective to delaypremature birth.
 47. The method of claim 46, wherein saidcytidine-containing compound, cytosine-containing compound,creatine-containing compound, uridine-containing compound,adenosine-containing compound, or adenosine-elevating compound, oromega-3 fatty acid is not administered in an amount effective to delaypremature birth.
 48. The method of claim 46, wherein said compound in(i) is a cytidine-containing compound, cytosine-containing compound, oruridine-containing compound.
 49. The method of claim 46, wherein saiduridine-containing compound is uridine, UTP, or triacetyl uridine. 50.The method of claim 46, wherein said cytidine-containing compound iscytidine or CDP.
 51. The method of claim 46, wherein saidcytidine-containing compound further comprises choline.
 52. The methodof claim 46, wherein said cytidine-containing compound is CDP-choline.53. The method of claim 46, wherein said omega-3 fatty acid iseicosapentaenoic acid, docosahexaenoic acid, or α-linolenic acid. 54.The method of claim 46, wherein said omega-3 fatty acid is administeredas fish oil or flaxseed oil.